Project Summary/Abstract In this revised R01 application we will ask how estrogen signaling in the central nervous system controls bone metabolism in a sex dependent manner. It is well known that peripheral estrogen affects bone in both mice and humans. As part of our efforts to understand how estrogen neural circuits in mice affect female metabolism, we discovered that estrogen signaling in the arcuate nucleus (ARC) of the hypothalamus normally suppresses cortical and trabecular bone mass in female mice. Ablating estrogen receptor alpha (ERa) in three intersectional and independent mouse models leads to a striking high bone mass phenotype in female mice; male bones are unaffected. Mutant females exhibit exceptionally dense trabecular and cortical bones, whose strengths surpass other reported mouse models. Stereotaxic- guided deletion of brain ERa confirmed the central origin of this bone phenotype and uncoupled this phenotype from changes in circulating estradiol, testosterone, or leptin. Our work defines central regulation of bone metabolism, alongside reproduction and energy balance, as a fundamental sex- difference in physiology. These data also offer proof for the concept that the brain is a major determinant of female bone metabolism, thus expanding the gatekeeper function of the hypothalamus in energy expenditure to non-classical metabolic tissues, such as bone. Although our research is in the earliest pre-clinical stages, understanding how our models build massively dense and strong bones could transform current rational design of therapeutics for hormonal and age-related bone loss. Why and how female ARC neurons normally inhibit bone metabolism remain a mystery. In the following three aims, we seek to unravel this puzzle. We will define and then confirm which ARC neurons mediate this brain-bone connection, ask if manipulating these neurons via chemogenetics and pharmacology mimics high bone phenotype or normalizes the bone phenotype in mutants, and then begin determining the molecular basis for this sex-dependent high bone phenotype.